Recombinant fusion proteins to growth hormone and serum albumin

ABSTRACT

Fusion proteins of albumin and growth hormone, or fusions of variants of either, are secreted well in yeast and have increased serum and storage stability.

FIELD OF THE INVENTION

[0001] The present invention relates to recombinant fusion proteins, togrowth hormone (GH), to serum albumin and to production of proteins inyeast.

BACKGROUND AND PRIOR ART

[0002] Human serum albumin (HSA), a protein of 585 amino acids, isresponsible for a significant proportion of the osmotic pressure ofserum and also functions as a carrier of endogenous and exogenousligands. At present, HSA for clinical use is produced by extraction fromhuman blood. The production of recombinant HA (rHA) in microorganismshas been disclosed in EP 330 451 and EP 361 991.

[0003] The role of albumin as a carrier molecule and its inert natureare desirable properties for use as a stabiliser and transporter ofpolypeptides. The use of albumin as a component of a fusion protein forstabilising other proteins has been disclosed in WO 93/15199, WO93/15200, and EP 413 622. The use of N-terminal fragments of HSA forfusions to polypeptides has also been disclosed (EP 399 666). Fusion tothe said polypeptide is achieved by genetic manipulation, such that theDNA coding for HSA, or a fragment thereof, is joined to the DNA codingfor the said polypeptide. A suitable host is then transformed ortransfected with the fused nucleotide sequences, so arranged on asuitable plasmid as to express a fusion polypeptide. Nomura et al (1995)attempted to express human apolipoprotein E in S. cerevistae as a fusionprotein with HSA or fragments of HSA, using the HSA pre-sequence todirect secretion. Whilst fusion to full length HSA resulted in thesecretion of low levels of the protein into the medium (maximum yield of6.3 mg per liter), fusion to HSA (1-198) or HSA (1-390) did not resultin secretion into the medium.

[0004] Human growth hormone (reviewed by Strobl and Thomas, 1994)consists of a single polypeptide of 191 amino acids, internallycross-linked by two disulphide bonds. Two molecules of hGH receptor bindeach molecule of hGH to facilitate signal transduction (Cunningham etal, 1991; de Vos et al, 1992). The C-terminus of the hGH molecule isinvolved in binding to the first receptor molecule, but the extent towhich the N-terminus is involved in receptor binding is not known. Thehormone is secreted from the anterior pituitary gland under hypothalamiccontrol, and is responsible for a wide range of growth-promoting effectsin the body. Clinically, hGH is used in the treatment of hypopituitarydwarfism, chronic renal insufficiency in childhood, bone fractures andburns. Current methods of production of hGH for therapeutic use are byextraction from human pituitary gland, recombinant expression inEscherichia coli as disclosed in EP 127 305 (Genentech) or recombinantexpression in mammalian cell culture (Zeisel et al, 1992).

[0005] In addition, hGH has been expressed intracellularly in yeast(Tokunaga et at, 1985) and this organism may provide an alternativemeans of production as disclosed in EP 60 057 (Genentech). Tsiomenko etal (1994) reported the role of the yeast MFα-1 prepro leader sequence inthe secretion of hGH from yeast. Attachment of the pre-portion of theleader sequence to the hGH gene resulted in hGH accumulation in theperiplasm and vacuoles, whilst attachment of the pro-portion to hGHresulted in expression of a non-glycosylated precursor localised insidethe cell. Only when both portions of the leader sequence were attachedto the hGH gene was hGH secreted into the culture medium. Othersecretion signals (pre-sequences) were also ineffective unless ayeast-derived pro sequence was used, suggesting that such a pro sequencewas used is critical to the efficient secretion of hGH in yeast.

[0006] In humans, hGH is secreted into the blood in pulses, and in thecirculation has a half-life of less than 20 minutes (Haffner et al,1994). Elimination of the hormone is primarily via metabolism in theliver and kidneys and is more rapid in adults than in children (Kearnset al, 1991). Treatment for hGH deficiency generally lasts for 6 to 24months, during which hGH is administered either three times a weekintramuscularly or on a daily basis subcutaneously. Such a regimen offrequent administration is necessary because of the short half-life ofthe molecule.

[0007] Poznansky et al (1988) increased the half-life of porcine growthhormone by conjugation with either porcine or human serum albumin (HSA)to form relatively large conjugates of about 180 kD. Chemical reactionusing the cross-linking reagent glutaraldehyde resulted in, on average,two molecules of albumin complexed with six molecules of growth hormone.The resulting 180 kD conjugate was found to have an extended half-lifein the circulation of rats of 2 to 3 hours, compared to 5 minutes forunconjugated growth hormone. Activity assays showed that the conjugateretained full, and possibly increased activity in vitro, but wasinactive in vivo.

SUMMARY OF THE INVENTION

[0008] The invention relates to proteins formed by the fusion of amolecule of albumin, or variants or fragments thereof, to a molecule ofgrowth hormone or variants or fragments thereof, the fusion proteinshaving an increased circulatory half-life over unfused growth hormone.For convenience, we shall refer to human albumin (HA) and human growthhormone (hGH), but the albumin and growth hormones of other vertebratesare included also. Preferably, the fusion protein comprises HA, or avariant or fragment thereof, as the N-terminal portion, with hGH or avariant or fragment thereof as the C-terminal portion, so as to minimiseany possible negative effects on receptor binding. Alternatively, afusion protein comprising HA, or a variant or fragment thereof, as theC-terminal portion, with hGH or a variant or fragment thereof as theN-terminal portion, may also be capable of signal transduction.Generally, the polypeptide has only one HA-derived region and oneGH-derived region.

[0009] Additionally, the fusion proteins of the invention may include alinker peptide between the two fused portions to provide a greaterphysical separation between the two moieties and thus maximise theavailability of the hGH portion to bind the hGH receptor. The linkerpeptide may consist of amino acids such that it is flexible or morerigid.

[0010] The linker sequence may be cleavable by a protease or chemicallyto yield the growth hormone related moiety. Preferably, the protease isone which is produced naturally by the host, for example the S.cerevisiae protease kex2 or equivalent proteases. Hence, a furtheraspect of the invention provides a process for preparing growth hormoneor a variant or fragment thereof by expressing a polynucleotide whichencodes a polypeptide of the invention in a suitable host, cleaving thecleavable linker to yield the GH-type compound and recovering theGH-type compound from the host culture in a more pure form.

[0011] We have discovered that the polypeptides of the invention aresignificantly more stable in solution than hGH. The latter rapidlybecomes inactive when stored in solution at 4° C. for over one month.Currently marketed hGH is sold as a freeze-dried powder.

[0012] Suitably, the fusion polypeptides are produced as recombinantmolecules by secretion from yeast, a microorganism such as a bacterium,human cell line or a yeast. Preferably, the polypeptide is secreted fromthe host. We have found that, by fusing the hGH coding sequence to theHA coding sequence, either to the 5′ end or 3′ end, it is possible tosecrete the fusion protein from yeast without the requirement for ayeast-derived pro sequence. This was surprising, as other workers havefound that a yeast derived pro sequence was needed for efficientsecretion of hGH in yeast. For example, Hiramitsu et al (1990, 1991)found that the N-terminal portion of the pro sequence in the Mucorpusillus rennin pre-pro leader was important. Other authors, using theMFα-1 signal, have always included the MFα-1 pro sequence when secretinghGH. The pro sequences were believed to assist in the folding of the hGHby acting as an intramolecular chaperone. The present invention showsthat HA or fragments of HA can perform a similar function.

[0013] Hence, a particular embodiment of the invention comprises a DNAconstruct encoding a signal sequence effective for directing secretionin yeast, particularly a yeast-derived signal sequence (especially onewhich is homologous to the yeast host), and the fused molecule of thefirst aspect of the invention, there being no yeast-derived pro sequencebetween the signal and the mature polypeptide.

[0014] The Saccharomyces cerevisiae invertase signal is a preferredexample of a yeast-derived signal.

[0015] Conjugates of the kind prepared by Poznansky et al (1988). inwhich separately-prepared polypeptides are joined by chemicalcross-inking, are not contemplated

[0016] The albumin or hGH may be a variant of normal HSA/rHA (termedhereinafter “HA”) or hGH, respectively. By “variants” we includeinsertions, deletions and substitutions, either conservative ornon-conservative, where such changes do not substantially alter one ormore of the oncotic, useful ligand-binding and non-immunogenicproperties of albumin or, in the case of hGH, its non-immunogenicity andability to bind and activate the hGH receptor. In particular, we includenaturally-occurring polymorphic variants of human albumin and fragmentsof human albumin, for example those fragments disclosed in EP 322 094(namely HA (1-n), where n is 369 to 419). The albumin or growth hormonemay be from any vertebrate, especially any mammal, for example human,cow, sheep, pig, hen or salmon. The albumin and GH parts of the fusionmay be from differing animals.

[0017] By “conservative substitutions” is intended swaps within groupssuch as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg;and Phe, Tyr. The variant will usually have at least 75% (preferably atleast 80%, 90%, 95% or 99%) sequence identity with a length of normal HAor hGH which is the same length as the variant and which is moreidentical thereto than any other length of normal HA or hGH, once theallowance is made for deletions and insertions as is customary in thisart. Generally speaking, an HA variant will be at least 100 amino acidslong, preferably at least 150 amino acids long. The HA variant mayconsist of or comprise at least one whole domain of HA, for exampledomains 1 (1-194), 2 (195-387), 3 (388-585), 1+2 (1-387), 2+3 (195-585)or 1+3 (1-194, +388-585). Each domain is itself made up of twohomologous subdomains namely 1-105 120-194, 195-291, 316-387, 388-491and 512-585, with flexible inter-subdomain linker regions comprisingresidues Lys106 to Glu199, Glu292 to Val315 and Glu492 to Ala511.Preferably, the HA part of the fusion comprises at least one subdomainor domain of HA or conservative modifications thereof. If the fusion isbased on subdomains, some or all of the adjacent linker is preferablyused to link to the hGH moiety. The hGH variant should have GH activity,and will generally have at least 10 amino acids, (although some authorshave found activity with only 4 residues), preferably at least 20,preferably at least 50, 100, 150, 180 or 191, amino acids long, andpreferably retains its cysteines for both internal disulphide bonds.

[0018] The fused molecules of the invention generally have a molecularweight of less than 100 kD, for example less than 90 kD or 70 kD. Theyare therefore much smaller than the 180 kD conjugates of Poznansky et al(referred to above), which were inactive in vivo. They will normallyhave a molecular weight of at least 20 kD, usually at least 30 kD or 50kD. Most fall within the molecular weight range 60-90 kD.

[0019] A second main aspect of the invention provides a yeasttransformed to express a fusion protein of the invention.

[0020] In addition to the transformed host cells themselves, the presentinvention also contemplates a culture of those cells, preferably amonoclonal (clonally homogeneous) culture, or a culture derived from amonoclonal culture, in a nutrient medium. Especially if the polypeptideis secreted, the medium will thus contain the polypeptide, with thecells, or without the cells if they have been filtered or centrifugedaway

[0021] Many expression systems are known, including bacteria (forexample E. coli and Bacillus subtilis), yeasts (for exampleSaccharomyces cerevisiae, Kluyveronmyces lactis and Pichia pastoris,filamentous fungi (for example Aspergillus), plant cells, animal cellsand insect cells.

[0022] The desired protein is produced in conventional ways, for examplefrom a coding sequence inserted in the host chromosome or on a freeplasmid.

[0023] The yeasts are transformed with a coding sequence for the desiredprotein in any of the usual ways, for example electroporation. Methodsfor transformation of yeast by electroporation are disclosed in Becker &Guarente (1990) Methods Enzymol. 194, 182.

[0024] Successfully transformed cells, ie cells that contain a DNAconstruct of the present invention, can be identified by well knowntechniques. For example, cells resulting from the introduction of anexpression construct can be grown to produce the desired polypeptide.Cells can be harvested and lysed and their DNA content examined for thepresence of the DNA using a method such as that described by Southern(1975) J. Mol. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208.Alternatively, the presence of the protein in the supernatant can bedetected using antibodies.

[0025] Useful yeast plasmid vectors include pRS403-406 and pRS413-416and are generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are YeastIntegrating plasmids (YIps) and incorporate the yeast selectable markersHIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromereplasmids (YCps).

[0026] A variety of methods have been developed to operably link DNA tovectors via complementary cohesive termini For instance, complementaryhomopolymer tracts can be added to the DNA segment to be inserted to thevector DNA. The vector and DNA segment are then joined by hydrogenbonding between the complementary homopolymeric tails to formrecombinant DNA molecules.

[0027] Synthetic linkers containing one or more restriction sitesprovide an alternative method of joining the DNA segment to vectors. TheDNA segment, generated by endonuclease restriction digestion, is treatedwith bacteriophage T4 DNA polymerase or E. coli DNA polymerase I,enzymes that remove protruding, 3′-single-stranded termini with their3′-5′-exonucleolytic activities, and fill in recessed 3′-ends with theirpolymerizing activities.

[0028] The combination of these activities therefore generatesblunt-ended DNA segments. The blunt-ended segments are then incubatedwith a large molar excess of linker molecules in the presence of anenzyme that is able to catalyze the ligation of blunt-ended DNAmolecules, such as bacteriophage T4 DNA ligase. Thus, the products ofthe reaction are DNA segments carrying polymeric linker sequences attheir ends. These DNA segments are then cleaved with the appropriaterestriction enzyme and ligated to an expression vector that has beencleaved with an enzyme that produces termini compatible with those ofthe DNA segment.

[0029] Synthetic linkers containing a variety of restrictionendonuclease sites are commercially available from a number of sourcesincluding International Biotechnologies Inc, New Haven, Conn., USA.

[0030] A desirable way to modify the DNA in accordance with theinvention, if, for example, HA variants are to be prepared, is to usethe polymerase chain reaction as disclosed by Saiki et al (1988) Science239, 487-491. In this method the DNA to be enzymatically amplified isflanked by two specific oligonucleotide primers which themselves becomeincorporated into the amplified DNA. The said specific primers maycontain restriction endonuclease recognition sites which can be used forcloning into expression vectors using methods known in the art.

[0031] Exemplary genera of yeast contemplated to be useful in thepractice of the present invention as hosts for expressing the fusionproteins are Pichia (Hansenula), Saccharomyces, Kluyveromyces, Candida,Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen,Debaromyces, Metschunikowia, Rhodosporidium, Leucosporidium,Botryoascus, Sporidiobolus, Endomycopsis, and the like. Preferred generaare those selected from the group consisting of Saccharomyces,Schizosaccharomyces, Kluyveromyces, Pichia and Torulaspora. Examples ofSaccharomyces spp. are S. cerevisiae, S. italicus and S. rouxii.Examples of Kluyveromyces spp are K. fragilis, K. lactis and K.marxianus. A suitable Torulaspora species is T. delbrueckii. Examples ofPichia (Hansenula) spp. are P. angusta (formerly H. polymorpha), P.anomala (formerly H. anomala) and P. pastoris.

[0032] Methods for the transformation of S. cerevisiae are taughtgenerally in EP 251 744, EP 258 067 and WO 90/01063, all of which areincorporated herein by reference.

[0033] Suitable promoters for S. cerevisiae include those associatedwith the PGK1 gene, GAL1 or GAL10 genes, CYC1, PHO5, TRP1, ADH1, ADH2,the genes for glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, triose phosphate isomerase,phosphoglucose isomerase, glucokinase, α-mating factor pheromone,a-mating factor pheromone, the PRB1 promoter the GUT2 promoter, the GPD1promoter and hybrid promoters involving hybrids of parts of 5′regulatory regions with parts of 5′ regulatory regions of otherpromoters or with upstream activation sites (eg the promoter of EP-A-258067).

[0034] Convenient regulatable promoters for use in Schizosaccharomycespombe are the thiamine-repressible promoter from the nmt gene asdescribed by Maundrell (1990) J. Biol. Chem. 265, 10857-10864 and theglucose-repressible fbp1 gene promoter as described by Hoffman & Winston(1990) Genetics 124, 807-816.

[0035] Methods of transforming Pichia for expression of foreign genesare taught in, for example, Cregg et al (1993), and various Phillipspatents (eg U.S. Pat. No. 4,857,467, incorporated herein by reference),and Pichia expression kits are commercially available from InvitrogenBV, Leek, Netherlands, and Invitrogen Corp., San Diego, Calif. Suitablepromoters include AOX1 and AOX2.

[0036] Gleeson et al (1986) J. Gen. Microbiol. 132, 3459-3465 includeinformation on Hansenula vectors and transformation, suitable promotersbeing MOX1 and FMD1; whilst EP 361 991, Fleer et al (1991) and otherpublications from Rhône-Poulenc Rorer teach how to express foreignproteins in Kluyveromyces spp., a suitable promoter being PGK1.

[0037] The transcription termination signal is preferably the 3′flanking sequence of a eukaryotic gene which contains proper signals fortranscription termination and polyadenylation. Suitable 3′ flankingsequences may, for example, be those of the gene naturally linked to theexpression control sequence used, ie may correspond to the promoter.Alternatively, they may be different in which case the terminationsignal of the S. cerevisiae ADH1 gene is preferred.

[0038] The desired fusion protein may be initially expressed with asecretion leader sequence, which may be any leader effective in theyeast chosen. Leaders useful in S. cerevisiae include that from themating factor α polypeptide (MFα-1) and the hybrid leaders of EP-A-387319. Such leaders (or signals) are cleaved by the yeast before themature albumin is released into the surrounding medium. Further suchleaders include those of S. cerevisiae invertase (SUC2) disclosed in JP62-096086 (granted as 91/036516), acid phosphatase (PH05), thepre-sequence of MFα-1,β-glucanase (BGL2) and killer toxin; S.diastaticus glucoamylase II; S. carlsbergensis α-galactosidase (MEL1);K. lactis killer toxin; and Candida glucoamylase.

[0039] The fusion protein of the invention or a formulation thereof maybe administered by any conventional method including parenteral (egsubcutaneous or intramuscular) injection or intravenous infusion. Thetreatment may consist of a single dose or a plurality of doses over aperiod of time.

[0040] Whilst it is possible for a fusion protein of the invention to beadministered alone, it is preferable to present it as a pharmaceuticalformulation, together with one or more acceptable carriers. Thecarrier(s) must be “acceptable” in the sense of being compatible withthe fusion protein and not deleterious to the recipients thereof.Typically, the carriers will be water or saline which will be sterileand pyrogen free. The formulation should be non-immunogenic;vaccine-type formulations involving adjuvants are not contemplated.

[0041] The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. Such methods include the step of bringing into association thefusion protein with the carrier which constitutes one or more accessoryingredients. In general the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product

[0042] Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example seated ampoules andvials, and may be stored in a freeze-dried (lyophilised) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders.

[0043] Preferred unit dosage formulations are those containing a dailydose or unit, daily sub-dose or an appropriate fraction thereof, of anactive ingredient.

[0044] The fusion proteins of the invention may be used in the treatmentof any condition in which growth hormone is indicated, for exampleisolated growth hormone deficiency, panhypopituitarism, followingcranial irradiation (eg in the treatment of leukaemia or brain tumours),Turner's syndrome, Down's syndrome, intrauterine growth retardation,idiopathic growth deficiency, chronic renal failure, achondroplasia,female infertility and various catabolic disorders. They may also beused in the stimulation of growth, and/or enhancement of lean meatproportion, in farm animals such as cows, sheep, goats and pigs.

[0045] The fusion protein may be administered together with insulin-likegrowth factor I (IGF-I).

[0046] The dosage can be calculated on the basis of the potency of thefusion protein relative to the potency of hGH, whilst taking intoaccount the prolonged serum half-life of the fusion proteins compared tothat of native hGH. Growth hormone is typically administered at 0.3 to30.0 IU/kg/week, for example 0.9 to 12.0 IU/kg/week, given in three orseven divided doses for a year or more. In a fusion protein consistingof full length HA fused to full length GH, an equivalent dose in termsof units would represent a greater weight of agent but the dosagefrequency can be reduced, for example to twice a week, once a week orless.

[0047] Preferred examples of the invention will now be described by wayof example and with reference to the accompanying figures, in which:

[0048]FIG. 1 shows the human growth hormone cDNA sequence, encodingmature hGH;

[0049]FIG. 2 shows a restriction enzyme map of pHGH1;

[0050]FIG. 3 shows a restriction enzyme map of pBST(+) and the DNAsequence of the polylinker;

[0051]FIG. 4 shows the construction of pHGH12,

[0052]FIG. 5 shows the construction of pHGH16;

[0053]FIG. 6 shows the HSA cDNA sequence, more particularly the regionencoding the mature protein;

[0054]FIG. 7 shows the construction of pHGH14;

[0055]FIG. 8 shows the construction of pHGH38;

[0056]FIG. 9 shows the construction of pHGH31;

[0057]FIG. 10 shows the construction of pHGH58 or pHGH59 (Example 7);

[0058]FIG. 11 is a scheme for constructing fusions having spacers(Example 7); and

[0059]FIG. 12 shows the results of a pharmacokinetic study showing theclearance of ¹²⁵I-labelled rHA-hGH compared to that of hGH following ivinjection in rats. Data are from two rats in each group, and includetotal radioactivity and radioactivity which could be precipitated byTCA, ie that associated with protein rather than as free ¹²⁵I. Thecalculated clearance half-life for hGH was approximately 6 minutes,compared to approximately 60 minutes for the rHA-hGH fusion protein. SeeExample 3.

[0060] —hGH (total counts)

[0061] ▪—hGH (TCA precipitated counts)

[0062] ▾—rHA-hGH (total counts)

[0063] ▴—rHA-hGH (TCA precipitated counts).

DETAILED DESCRIPTION OF THE INVENTION

[0064] All standard recombinant DNA procedures are as described inSambrook et al (1989) unless otherwise stated. The DNA sequencesencoding HSA were derived from the cDNA disclosed in EP 201 239.

EXAMPLE 1 Cloning of the hGH cDNA

[0065] The hGH cDNA was obtained from a human pituitary gland cDNAlibrary (catalogue number HL1097v, Clontech Laboratories, Inc) by PCRamplification. Two oligonucleotides suitable for PCR amplification ofthe hGH cDNA, HGH1 and HGH2, were synthesised using an AppliedBiosystems 380B Oligonucleotide Synthesiser. HGH1:5′-CCCAAGAATTCCCTTATCCAGGC-3′ HGH2:5′-GGGAAGCTTAGAAGCCACAGGATCCCTCCACAG-3′

[0066] HGH1 and HGH2 differed from the equivalent portion of the hGHcDNA sequence (FIG. 1, Martial et al, 1979) by two and threenucleotides, respectively, such that after PCR amplification an EcoRIsite would be introduced to the 5′ end of the cDNA and a BamHI sitewould be introduced into the 3′ end of the cDNA. In addition, HGH2contained a HindIII site immediately downstream of the hGH sequence.

[0067] PCR amplification using a Perkin-Elmer-Cetus Thermal Cycler 9600and a Perkin-Elmer-Cetus PCR kit, was performed using single-strandedDNA template isolated from the phage particles of the cDNA library asfollows: 10 μL phage particles were lysed by the addition of 10 μL phagelysis buffer (280 μg/mL proteinase K in TE buffer) and incubation at 55°C. for 15 min followed by 85° C. for 15 min After a 1 min incubation onice, phage debris was pelleted by centriftigation at 14,000 rpm for 3min. The PCR mixture contained 6 μL of this DNA template, 0.1 μM of eachprimer and 200 μM of each deoxyribonucleotide. PCR was carried out for30 cycles, denaturing at 94° C. for 30 s, annealing at 65° C. for 30 sand extending at 72° C. for 30 s, increasing the extension time by 1 sper cycle. Analysis of the reaction by gel electrophoresis showed asingle product of the expected size (589 base pairs).

[0068] The PCR product was purified using Wizard PCR Preps DNAPurification System (Promega Corp) and then digested with EcoRI andHindIII. After further purification of the EcoRI-HindIII fragment by gelelectrophoresis, the product was cloned into pUC19 (GIBCO BRL) digestedwith EcoRI and HindIII, to give pHGH1 (FIG. 2). DNA sequencing of theEcoRI-HindIII region showed that the PCR product was identical insequence to the hGH sequence (Martial et al, 1979), except at the 5′ and3′ ends, where the EcoRI and BamHI sites had been introduced,respectively.

EXAMPLE 2 Expression of the hGH cDNA

[0069] The polylinker sequence of the phagemid pBluescribe (+)(Stratagene) was replaced by inserting an oligonucleotide linker, formedby annealing two 75-mer oligonucleotides, between the EcoRI and HindIIIsites to form pBST(+) (FIG. 3). The new polylinker included a uniqueNotI site (the full sequence in the region of the polylinker is given inFIG. 3).

[0070] The NotI HSA expression cassette of pAYE309 (EP 431 880)comprising the PRB1 promoter. DNA encoding the HSA/MFα-1 hybrid leadersequence, DNA encoding HSA and the ADH1 terminator, was transferred topBST(+) to form pHA1 (FIG. 4). The HSA coding sequence was removed fromthis plasmid by digestion with HindIII followed by religation to formpHA2 (FIG. 4).

[0071] Cloning of the hGH cDNA, as described in Example 1, provided thehGH coding region lacking the pro-hGH sequence and the first 8 basepairs (bp) of the mature hGH sequence. In order to construct anexpression plasmid for secretion of hGH from yeast, a yeast promoter,signal peptide and the first 8 bp of the hGH sequence were attached tothe 5′ end of the cloned hGH sequence as follows:

[0072] The HindIII-SfaNI fragment from pHA1 was attached to the 5′ endof the EcoRI-HindIII fragment from pHGH1 via two syntheticoligonucleotides, HGH3 and HGH4: HGH3: 5′-GATAAAGATTCCCAAC-3′ HGH4:5′-AATTGTTGGGAATCTTT-3′

[0073] The HindIII fragment so formed was cloned into HindIII-digestedpHA2 to make pHGH2 (FIG. 4), such that the hGH cDNA was positioneddownstream of the PRB1 promoter and HSA/MFα-1 fusion leader sequence (WO90/01063). The NotI expression cassette contained in pHGH2, whichincluded the ADH1 terminator downstream of the hGH cDNA, was cloned intoNotI-digested pSAC35 (Sleep et al, 1990) to make pHGH12 (FIG. 4). Thisplasmid comprised the entire 2 μm plasmid to provide replicationfunctions and the LEU2 gene for selection of transformants.

[0074] pHGH12 was introduced into S. cerevisiae DB1 (Sleep et al, 1990)by transformation and individual transformants were grown for 3 days at30° C. in 10 mL YEPD (1% w/v yeast extract, 2% w/v peptone, 2% w/vdextrose). After centrifugation of the cells, the supernatants wereexamined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and werefound to contain protein which was of the expected size and which wasrecognised by anti-hGH antiserum (Sigma, Poole, UK) on Western blots.

EXAMPLE 3 Cloning and Expression of an HSA-hGH Fusion Protein

[0075] In order to fuse the HSA cDNA to the 5′ end of the hGH cDNA, thepHA1 HindIII-Bsu36I fragment (containing most of the HSA cDNA) wasjoined to the pHGH1 EcoRI-HindIII fragment (containing most of the hGHcDNA) via two oligonucleotides, HGH7 and HGH8: HGH7:5′-TTAGGCTTATTCCCAAC-3′ HGH8: 5′-AATTGTTGGGAATAAGCC-3′

[0076] The HindIII fragment so formed was cloned into pHA2 digested withHindIII to make pHGH10 (FIG. 5), and the NotI expression cassette ofthis plasmid was cloned into NotI-digested pSAC35 to make pHGH16 (FIG.5).

[0077] pHGH16 was used to transform S. cerevisiae DB1 and supernatantsof cultures were analysed as in Example 2. A predominant band wasobserved that had a molecular weight of approximately 88 kD,corresponding to the combined masses of HA and hGH. Western blottingusing anti-HSA and anti-hGH antisera (Sigma) confirmed the presence ofthe two constituent parts of the fusion protein.

[0078] The fusion protein was purified from culture supernatant bycation exchange chromatography, followed by anion exchange and gelpermeation chromatography. Analysis of the N-terminus of the protein byamino acid sequencing confirmed the presence of the expected albuminsequence.

[0079] An in vitro growth hormone activity assay (Ealey et al, 1995)indicated that the fusion protein possessed full hGH activity, but thatthe potency was reduced compared to the hGH standard. In ahypophysectomised rat weight gain model, performed essentially asdescried in the European Pharmacopoeia (1987, monograph 556), the fusionmolecule was more potent than hGH when the same number of units ofactivity (based on the above in vitro assay) were administered daily.Further experiments in which the fusion protein was administered onceevery four days showed a similar overall growth response to a dailyadministration of hGH. Pharmacokinetic experiments in which¹²⁵I-labelled protein was administered to rats indicated anapproximately ten-fold increase in circulatory half life for the fusionprotein compared to hGH (FIG. 12).

[0080] A similar plasmid was constructed in which DNA encoding the S.cerevisiae invertase (SUC2) leader sequence replaced the sequence forthe hybrid leader, such that the encoded leader and the junction withthe HSA sequence were as follows: MLLQAFLFLLAGFAAKISA↓DARKS. . .    Invertase leader   HSA

[0081] On introduction into S. cerevisiae DB1, this plasmid directed theexpression and secretion of the fusion protein at a level similar tothat obtained with pHGH16. Analysis of the N-terminus of the fusionprotein indicated precise and efficient cleavage of the leader sequencefrom the mature protein.

EXAMPLE 4 Cloning and Expression of an hGH-HSA Fusion Protein

[0082] In order to fuse the hGH cDNA to the 5′ end of the HSA cDNA (FIG.6), the HSA cDNA was first altered by site-directed mutagenesis tointroduce an EcoNI site near the 5′ end of the coding region. This wasdone by the method of Kunkel et al (1987) using single-stranded DNAtemplate prepared from pHA1 and a synthetic oligonucleotide, LEU4:

[0083] LEU4: 5′-GAGATGCACACCTGAGTGAGG-3′

[0084] Site-directed mutagenesis using this oligonucleotide changed thecoding sequence of the HSA cDNA from Lys4 to Leu4 (K4L). However, thischange was repaired when the hGH cDNA was subsequently joined at the 5′end by linking the pHGH2 NotI-BamHI fragment to the EcoNI-NotI fragmentof the mutated pHA1, via the two oligonucleotides HGH5 and HGH6: HGH5:5′-GATCCTGTGGCTTCGATGCACACAAGA-3′ HGH6: 5′-CTCTTGTGTGCATCGAAGCCACAG-3′

[0085] The NotI fragment so formed was cloned into NotI-digested pSAC35to make pHGH14 (FIG. 7). pHGH14 was used to transform S. cerevisiae DB1and supernatants of cultures were analysed as in Example 2. Apredominant band was observed that had a molecular weight ofapproximately 88 kD, corresponding to the combined masses of hGH and HA.Western blotting using anti-HSA and anti-hGH antisera confirmed thepresence of the two constituent parts of the fusion protein.

[0086] The fusion protein was purified from culture supernatant bycation exchange chromatography, followed by anion exchange and gelpermeation chromatography. Analysis of the N-terminus of the protein byamino acid sequencing confirmed the presence of the expected hGHsequence.

[0087] In vitro studies showed that the fusion protein retained hGHactivity, but was significantly less potent than a fusion proteincomprising full-length HA (1-585) as the N-terminal portion and hGH asthe C-terminal portion, as described in Example 3.

EXAMPLE 5 Construction of Plasmids for the Expression of hGH Fusions toDomains of HSA

[0088] Fusion polypeptides were made in which the hGH molecule was fusedto the first two domains of HA (residues 1 to 387). Fusion to theN-terminus of hGH was achieved by joining the pHA1 HindIII-SapIfragment, which contained most of the coding sequence for domains 1 and2 of HA, to the pHGH1 EcoRI-HindIII fragment, via the oligonucleotidesHGH11 and HGH12: HGH11: 5′-TGTGGAAGAGCCTCAGAATTTATTCCCAAC-3′ HGH12:5′-AATTGTTGGGAATAAATTCTGAGGCTCTTCC-3′

[0089] The HindIII fragment so formed was cloned into HindIII-digestedpHA2 to make pHGH37 (FIG. 8) and the NotI expression cassette of thisplasmid was cloned into NotI-digested pSAC35. The resulting plasmid,pHGH38 (FIG. 8), contained an expression cassette that was found todirect secretion of the fusion polypeptide into the supernatant whentransformed into S. cerevisiae DB1. Western blotting using anti-HSA andanti-hGH antisera confirmed the presence of the two constituent parts ofthe fusion protein.

[0090] The fusion protein was purified from culture supernatant bycation exchange chromatography followed by gel permeationchromatography. In vivo studies with purified protein indicated that thecirculatory half-life was longer than that of hGH, and similar to thatof a fusion protein comprising full-length HA (1-585) as the N-terminalportion and hGH as the C-terminal portion, as described in Example 3. Invitro studies showed that the fusion protein retained hGH activity.

[0091] Using a similar strategy as detailed above, a fusion proteincomprising the first domain of HA (residues 1-194) as the N-terminalportion and hGH as the C-terminal portion, was cloned and expressed inS. cerevisiae DB1. Western blotting of culture supernatant usinganti-HSA and anti-hGH antisera confirmed'the presence of the twoconstituent parts of the fusion protein.

EXAMPLE 6 Expression of hGH by Introducing a Cleavage Site Between HSAand hGH

[0092] Introduction of a peptide sequence that is recognised by the Kex2protease, between the HA-hGH fusion protein, allows secretion of hGH. Asequence encoding Ser Leu Asp Lys Arg was introduced using twooligonucleotides, HGH14 and HGH15: HGH14:5′-TTAGGCTTAAGCTTGGATAAAAGATTCCCAAC-3′ HGH15:5′-AATTGTTGGGAATCTTTTATCCAAGCTTAAGCC-3′

[0093] These were used to join the pHA1 HindIII-Bsu36I fragment to thepHGH1 EcoRI-HindIII fragment, which were then cloned intoHindIII-digested pHA2 to make pHGH25 (FIG. 9) The NotI expressioncassette of this plasmid was cloned into NotI-digested pSAC35 to makepHGH31 (FIG. 9)

[0094]S. cerevisiae DBI transformed with pHGH31 was found to secrete twomajor species, as determined by SDS-PAGE analysis of culturesupernatants. The two species had molecular weights of approximately 66kD, corresponding to (full length) HA, and 22 kD, corresponding to (fulllength) hGH, indicating in vivo cleavage of the fusion protein by theKex2 protease, or an equivalent activity. Western blotting usinganti-HSA and anti-hGH antisera confirmed the presence of the twoseparate species. N-terminal sequence analysis of the hGH moietyconfirmed the precise and efficient cleavage from the HA moiety.

[0095] The hGH moiety was purified from culture supernatant by anionexchange chromatography followed by gel permeation chromatography. Invitro studies with the purified hGH showed that the protein was activeand fully potent.

[0096] Using a similar strategy, fusion proteins comprising either thefirst domain of HA (residues 1-194) or the first two domains of HA(residues 1-387), followed by a sequence recognised by the Kex2pprotease, followed by the hGH cDNA, were cloned and expressed in S.cerevisiae DB1. Western blotting of culture supernatant using anti-HSAand anti-hGH antisera confirmed the presence of the two separatespecies.

EXAMPLE 7 Fusion of HSA to hGH Using a Flexible Linker Sequence

[0097] Flexible linkers, comprising repeating units of[Gly-Gly-Gly-Gly-Ser]_(n), where n was either 2 or 3, were introducedbetween the HSA and hGH fusion protein by cloning of theoligonucleotides HGH16, HGH17, HGH18 and HGH19: HGH16:5′-TTAGGCTTAGGTGGCGGTGGATCCGGCGGTGGTGGATCTTT CCCAAC-3′ HGH17:5′-AATTGTTGGGAAAGATCCACCACCGCCGGATCCACCGCCAC CTAAGCC-3′ HGH18:5′-TTAGGCTTAGGCGGTGGTGGATCTGGTGGCGGCGGATCTGG TGGCGGTGGATCCTTCCCAAC-3′HGH19: 5′-AATTGTTGGGAAGGATCCACCGCCACCAGATCCGCCGCCAC CAGATCCACCACCGCCTAAGCC-3′

[0098] Annealing of HGH16 with HGH17 resulted in n=2, while HGH18annealed to HGH19 resulted in n=3. After annealing, the double-strandedoligonucleotides were cloned with the EcoRI-Bsu36I fragment isolatedfrom pHGH1 into Bsu36I-digested pHGH10 to make pHGH56 (where n=2) andpHGH57 (where n=3) (FIG. 10). The NotI expression cassettes from theseplasmids were cloned into NotI-digested pSAC35 to make pHGH58 andpHGH59, respectively.

[0099] Cloning of the oligonucleotides to make pHGH56 and pHGH57introduced a BamHI site in the linker sequences, as shown in FIG. 11. Itwas therefore possible to construct linker sequences in which n=1 andn=4, by joining either the HindIII-BamHI fragment from pHGH56 to theBamHI-HindIII fragment from pHGH57 (making n=1), or the HindIII-BamHIfragment from pHGH57 to the BamHI-HindIII fragment from pHGH56 (makingn=2). Cloning of these fragments into the HindIII site of pHA2(described in Example 2), resulted in pHGH60 (n=1) and pHGH61 (n4) (seeFIG. 11). The NotI expression cassettes from pHGH60 and pHGH61 werecloned into NotI-digested pSAC35 to make pHGH62 and pHGH63,respectively.

[0100] Transformation of S. cerevisiae with pHGH58. pHGH59, pHGH62 andpHGH63 resulted in transformants that secreted the fusion polypeptidesinto the supernatant.

[0101] Western blotting using anti-HSA and anti-hGH antisera confirmedthe presence of the two constituent parts of the fusion proteins.

[0102] The fusion proteins were purified from culture supernatant bycation exchange chromatography, followed by anion exchange and gelpermeation chromatography. Analysis of the N-termini of the proteins byamino acid sequencing confirmed the presence of the expected albuminsequence. Analysis of the purified proteins by electrospray massspectrometry confirmed an increase in mass of 315 D (n=1), 630 D (n=2),945 D (n=3) and 1260 D (n=4) compared to the HSA-hGH fusion proteindescribed in Example 3, as expected. The purified protein was found tobe active in vitro.

REFERENCES

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1. A polypeptide consisting of a continuous region of amino acids joinedtogether by peptide bonds and comprising a first region of at least 10amino acids which has at least 75% sequence identity with a same-lengthregion of growth hormone (GH) and a second region of at least 10 aminoacids which has at least 75% sequence identity with a same-length regionof serum albumin.
 2. A polypeptide according to claim 1 in which each ofsaid first and second regions has at least 95% sequence identity withthe said lengths of GH and albumin, respectively.
 3. A polypeptideaccording to claim 2 wherein each of said first and second regions is atleast 50 amino acids long.
 4. A polypeptide according to any one of thepreceding claims wherein the first region consists of uninterruptedamino acids of the C-terminus of GH, or a conservative modificationthereof, and the polypeptide binds and activates the GH receptor.
 5. Apolypeptide according to claim 4 wherein the first region is 191 aminoacids long.
 6. A polypeptide according to any one of the precedingclaims wherein the albumin and/or the growth hormone is human.
 7. Apolypeptide according to any one of the preceding claims wherein thesecond region comprises at least one uninterrupted domain of albumin ora conservative modification thereof.
 8. A polypeptide according to claim7 wherein the second region comprises uninterrupted amino acids 1-105,120-194, 195-291, 316-387, 388-491, 512-585, 1-194, 195-387, 388-585,1-387 or 195-585 of human albumin or a conservative modificationthereof.
 9. A polypeptide according to any one of the preceding claimswherein the N-terminus of the polypeptide comprises the said firstregion and the C-terminus comprises the said second region.
 10. Apolypeptide according to any one of claims 1 to 8 wherein the N-terminusof the polypeptide comprises the said second region and the C-terminuscomprises the said first region.
 11. A polypeptide according to any oneof the preceding claims which consists of the said first and secondregions, optionally with further amino acids or other compounds added toeither end of the polypeptide.
 12. A microbial culture medium comprisingtransformed cells and a polypeptide according to any one of thepreceding claims.
 13. A microbial culture medium comprising apolypeptide according to any one of claims 1 to
 11. 14. A polynucleotideencoding a polypeptide according to any one of claims 1 to
 11. 15. Amicrobial host comprising a polynucleotide according to claim 14arranged for expression in the host.
 16. A host according to claim 15wherein the polypeptide is secreted from the host.
 17. A process forobtaining a polypeptide according to any one of claims 1 to 11comprising culturing a host according to claim 15 or 16 and purifyingthe polypeptide.
 18. A pharmaceutical formulation comprising apolypeptide according to any one of claims 1 to 11 and apharmaceutically acceptable carrier.
 19. A method of treating a patienthaving a condition which is treatable with growth hormone, the methodcomprising administering to the patient a said-condition-alleviating,non-toxic amount of a polypeptide according to any one of claims 1 to11.
 20. A method of increasing beyond normal the growth rate of, orratio of lean meat to fat meat in, an animal comprising administering tothe animal an effective amount of a polypeptide according to any one ofclaims 1 to
 11. 21. A process for producing growth hormone, comprisingexpressing a polynucleotide according to claim 14 in a microbial host,preferably yeast, the polypeptide encoded thereby having a site betweenthe said first and second regions which site is cleavable by an enzymepresent in the host, or by any other enzyme, or chemically.